New species arise when populations evolve incompatible alleles that lead to lethality or sterility in hybrid offspring between isolated populations. Understanding the evolution of reproductive isolation between species requires functional characterization of the genes that cause barriers to genetic exchange and determining how they kill or sterilize hybrids. Two striking patterns in speciation biology are the early accumulation of hybrid male sterility in animal taxa with X and Y chromosomes (Haldane's Rule), and the enrichment of hybrid sterility factors on the X chromosome (the large X-effect). Speciation in XY species such as mammals and fruit flies is therefore associated with the rapid evolution of X-linked alleles that cause male sterility in interspecific hybrids. Multiple evolutionary models have been proposed to explain both these patterns, but there is insufficient empirical data to test these hypotheses. The long-term objectives of the proposed research are to identify previously unknown X-linked genes that cause hybrid male sterility between closely related species of Drosophila, and to characterize the mechanisms by which these genes disrupt spermatogenesis. These objectives will increase our understanding of the biological processes of speciation, evolutionary divergence in genes and genomes, and the developmental process of spermatogenesis. The proposed research will use genetic and transgenic approaches to identify, verify, and investigate X-linked hybrid sterility genes. Genome-wide gene expression and cytological analyses will determine how these genes disrupt spermatogenesis in hybrids; and population and evolutionary genomic resources will be used to determine the evolutionary history of these genes.